Interpretive Summary: The multi-laboratory broth microdilution method trial was performed to standardize specialized test conditions required for the bacterial fish pathogens Flavobacterium columnare and F. pyschrophilum. Consistent MIC results from these trials demonstrated that E.coli ATCC 25922 and A. salmonicida ATCC 33658 are reliable quality control strains for broth microdilution testing at 18°C and 28°C in diluted cation-adjusted Mueller Hinton broth. These strains can be used for QC testing of clinical isolates of F. columnare and F. pyschrophilum.

Technical Abstract:
A multi-laboratory broth microdilution method trial was performed to standardize the specialized test conditions required for fish pathogens Flavobacterium columnare and F. pyschrophilum. Nine laboratories tested the quality control (QC) strains Escherichia coli ATCC 25922 and Aeromonas salmonicida ATCC 33658 against 10 antimicrobials (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetorim/sulfadimethoxine, oxilinic acid, oxytetracycline, and trimethoprim/sulfamethoxazole) in diluted ( 4 g l-1) cation-adjusted Mueller-Hinton broth incubated at 28 and 18°C for 44-48 and 92-96 h, respectively. QC ranges were set for 9 of 10 antimicrobials. Most of the minimal inhibitory concentration (MIC) distributions (16 of 18, 9 drugs at both temperatures) for A. salmonicida ATCC 33658 were centered on a single median MIC + two-fold drug dilution resulting in a QC range that spanned 3 dilutions. More of the E. coli ATCC 25922 MIC distributions (7 of 16 ) were centered between 2 MIC dilutions requiring a QC range that spanned 4 dilutions. A QC range could not be determined for E. coli ATCC 25922 against 2 antimicrobials at the low temperature. These data and their associated QC ranges have been approved by the Clinical and Laboratory Standards Institute (CLSI) , and will be included in the next addition of the CLSI M49-A Guideline. This method represents the first standardized reference method for testing fish pathogenic Flavobacterium spp.